Liposomes are delivery vehicles prepared from aqueous dispersions of amphipathic lipids arranged in one or more bilayers around a central aqueous core. Solutes may be entrapped in the internal aqueous compartment thereby protecting them from reaction with blood components. In order to effectively deliver an entrapped agent to a target site, it is desirable that a liposome exhibits optimal circulation longevity and retention of an encapsulated agent.
The pharmacological properties of liposomes may be varied by changing the types of lipids incorporated in the membrane. Liposomes composed of neutral (no net charge) lipids are commonly employed as such liposomes are stable upon exposure to the numerous protein, carbohydrate and lipid components present in the blood compartment. Incorporating negatively charged lipids such as phosphatidylserine in liposomal compositions has resulted in increased recognition and clearance of liposomes from the circulation. Kirby, et al., Biochem. J. (1980) 186:591-598. Thus, drug delivery to disease sites is reduced. Allen, et al. Proc. Natl. Acad. Sci. USA (1998) 85:8061-8071.
A comparison of the circulation lifetimes of liposomes containing phosphatidylinositol, phosphatidylglycerol, cardiolipin and phosphatidylserine revealed that phosphatidylserine liposomes were eliminated quickly from the circulation whereas cardiolipin, phosphatidylglycerol and phosphatidylinositol liposomes were eliminated at a reduced rate in rats (Kao, Y, et al., J. Pharm. Sci. (1980) 69:1338-1349).
Although it has been acknowledged that certain negatively charged liposomes may have utility in in vivo applications, the use of these lipids to confer long-circulating properties has only recently been recognized. WO99/59547. These studies have revealed that the incorporation of phosphatidylglycerol into cholesterol/DPPC containing liposomes led to enhanced blood stability. These investigations also revealed, however, that the mol % incorporation of the lipid dimyristoylphosphatidylglycerol (DMPG) had an influence on the circulation properties of the carrier as cholesterol/DPPC liposomes prepared with less than 10 mol % DMPG exhibited increased blood stability properties in relation to those prepared with greater than 10 mol % DMPG. It was also noted that lipid components such as distearoylphosphatidylcholine (DSPC) and dimyristoyl-phosphatiylcholine (DMPC) possess undesirable properties and thus were not included in these formulations.
Liposomal preparations which contain phosphatidyl glycerol in addition to additional vesicle-forming lipids are described in Brodt, P., et al., Cancer Immunol. Immunother. (1989) 28:54-58 and by Hope, et al., U.S. Pat. No. 6,139,871. In these preparations, however, the negatively charged lipid coupled to a hydrophilic portion which is non-zwitterionic, exemplified by PG, is present in an amount less than 10 mol %. Similarly, the liposomes described in WO99/59547 include less than 10 mol % PG. The liposomes described by Akhtar, S., et al., Nucleic Acids Res. (1991) 19:5551-5559 and by Ahl, P. L., et al., Biochim. Biophys. Acta (1997) 1329:370-382 and by Farmer, M. C., et al., Meth. Enzymol. (1987) 149:184-200 contain substantial amounts of cholesterol, unlike the liposomes of the present invention. In addition, the liposomes of Akhtar exhibit transition temperatures lower than 38° C. as the acyl chains contained in the phospholipids employed contain less than 18 carbon atoms.
U.S. Pat. No. 5,415,869 describes taxol formulations contained in liposomes which may contain phosphatidylcholines and phosphatidyl glycerols. There is no suggestion that the liposomes have extended circulation lives, nor is it indicated that transition temperatures above 38° C. are preferred.
U.S. Pat. No. 4,769,250 describes formulations made from mixtures of anionic and neutral phospholipids including DSPG and DSPC; however, these liposomes are SUVs with dimensions of 45-55 nm.
Cryoprotectants have been added to liposome preparations in order to prevent the detrimental effects due to freezing and freeze-drying (lyophilization). Disaccharides such as trehalose, sucrose, lactose, sorbitol, mannitol, sucrose, maltodextrin and dextran are the most commonly used cryoprotectants (see WO01/05372 and U.S. Pat. No. 5,077,056).
Membrane-bound cryoprotectants have been utilized with the goal of further improving resistance to freezing and freeze-drying damage. In particular, sugars attached to liposomal membrane surfaces via oligo(ethylene oxide) linkers consisting of one to three repeating units have been reported to be cryoprotective (Bendas, et al., Eur. J. Pharma. Sci. (1996) 4:211-222; Goodrich, et al., Biochemistry (1991) 30:5313-5318; U.S. Pat. No. 4,915,951). Baldeschwieler, et al., reported that in the absence of the terminal sugar group, liposomes prepared with the oligoethylene oxide linker itself were unable to protect against fusion subsequent to freezing (see U.S. Pat. No. 4,915,951).
Inclusion of cholesterol in liposomal membranes has been shown to reduce release of drug after intravenous administration (for example, see: U.S. Pat. Nos. 4,756,910, 5,077,056, and 5,225,212; Kirby, C., et al., Biochem. J. (1980) 186:591-598; and, Ogihara-Umeda, I., et al., Eur. J. Nucl. Med. (1989) 15:617). Generally, cholesterol increases bilayer thickness and fluidity while decreasing membrane permeability, protein interactions, and lipoprotein destabilization of the liposome. Conventional approaches to liposome formulation dictate inclusion of substantial amounts (e.g., 30-45 mol %) cholesterol or equivalent membrane rigidification agents (such as other sterols) into liposomes.